1. Field of the Invention
The present invention relates generally to the fields of radiation biology and cell biology. More particularly, it concerns the attenuation of the effect of ionizing radiation induced activation of tumor necrosis factor by inhibitors of extranuclear signal transduction.
2. Description of the Related Art
Signaling pathways activated by DNA damage contribute to survival of prokaryotes and eukaryotic cells following exposure to x-rays or UV light. In irradiated E. coli, damaged DNA forms a complex with the Rec A protease resulting in the transcriptional induction of a variety of genes including those encoding DNA repair enzymes (Walker, 1985). In yeast, UV light and x-rays result in the induction of genes which participate in the repair of damaged DNA (Jones et al., 1991, Cole et al., 1987). Genes whose products are proposed to recognize damaged or un-replicated DNA and to participate in intracellular signaling that regulates cell cycle progression and DNA repair have been identified in S. cerevisiae and S. pombe (House et al., 1992, Enoch et al., 1992). The complexity of this signaling pathway is demonstrated by the number of genes involved in sensing DNA damage and transmitting the signal (Enoch et al., 1992). DNA damage is presumed to be the initiating event in mammalian cell induction of stress response genes following x-ray or UV exposure (Herrlich et al., 1992, Kastan et al., 1992). However, the mechanisms of DNA damage recognition have not been identified in mammalian cells.
Signal transduction pathways activated by ionizing radiation include increased phosphotransferase activity of cytoplasmic protein kinases (Hallahan et al., 1991a, Hallahan et al., 1991b, Uckun et al., 1992). Moreover, inhibition of protein kinases blocks radiation-mediated gene induction and effects diverse biological endpoints such as apoptosis (Uckun et al., 1992), radiation survival (Hallahan et al., 1992)) and induction of the cytokine tumor necrosis factor (TNF) (Hallahan et al., 1991b). The calcium/phospholipid-dependent protein kinase (PKC) is activated within 15 seconds of ionizing radiation exposure and is extinguished by 90 seconds in human leukemia HL-60 cells (Hallahan- et al., 1991b).
Phospholipase A2 inhibitors used in clinical radiotherapy to ameliorate acute and subacute sequelae include glucocorticoids and pentoxifylline,(Bianco et al., 1991, Phillips et al., 1975). Glucocorticoids are used to treat radiation induced proctitis, pneumonitis, conjunctivitis, external otitis, CNS syndromes and occasionally mucositis. Pentoxifylline is effective in preventing pneumonitis and mucositis following total body irradiation prior to bone marrow transplantation (Bianco et al., 1991). Taken together, these findings implicate phospholipase A2 in radiation induced TNF induction and the acute sequelae of radiotherapy.
Since phospholipase A2 hydrolyses phosphatidylcholine to arachidonic acid, the effects of the phospholipase A2 inhibitors mepacrine (Rao et al., 1993), and bromphenylbromide (BPB) (Peppelenbosch et al., 1993) were investigated. In addition, the effects of dexamethasone and pentoxifylline on radiation-induced fatty acid hydrolysis were studied, as these agents have been shown to inhibit phospholipase A2, reduce the production of cellular mediators of inflammation and tissue injury, and inhibit lipopolysaccharide-induced TNF production in monocytes (Strieter et al., 1988, Han et al., 1990). Moreover, glucocorticoids and pentoxifylline are employed clinically to prevent some acute toxicities of radiotherapy (Bianco et al., 1991, Phillips et al., 1975). The inventors determined that each agent attenuated arachidonic acid release into the medium of cells treated with X-rays or H2O2. Thus, extranuclear second messengers are in part responsible for radiation-mediated signal transduction and inhibition of this pathway may provide a means of attenuating the inflammatory-like response observed in irradiated tissues through the inhibition of TNF gene induction.
The present invention, in a general and overall sense, concerns methods of inhibiting the production of cytokines, for example tumor necrosis factor (TNF), following exposure of cells to ionizing radiation. In accordance with these methods, cells or tissues are contacted with phospholipase A2 inhibitors prior to exposure to ionizing radiation, reducing the production of cellular mediators of inflammation and tissue injury, and inhibiting the radiation-induced TNF production.
As used herein, xe2x80x9ccytokinexe2x80x9drefers to a class of molecules that are secreted by cells that affect the functions of other cells. More specifically, the cytokines of the present invention are secreted in response to ionizing radiation, and are produced as a result of or are otherwise involved in the arachidonic acid metabolic pathway.
Preferred inhibitors of phospholipase A2 include, but are not limited to mepacrine, bromphenylbromide (BPB), dexamethasone, or pentoxifylline. It will be recognized by-those skilled in the art that compounds with similar activity or derivatives of these inhibitors does not depart from the scope or spirit of the invention.
The present invention thus encompasses the use of any phospholipase inhibitor derivative that has a significant (i.e., consistently above background) inhibitory effect on phospholipase activity.
In certain embodiments of the invention, methods are provided for the treatment of acute radiation sequelae that mimic local inflammatory reactions, such as pneumonitis, proctitis, mucositis, dermatitis, and esophagitis. These consequences of radiotherapy may be associated with cytokine production, such as tumor necrosis factor, or with arachidonic acid metabolites. The methods described herein are designed to ameliorate these acute side effects associated with radiation therapy.
On other embodiments, the methods of the present invention are useful in inhibiting cytokine production in vitro following ionizing radiation exposure. The methods allow the production of certain polypeptides operatively linked to radiation inducible promoters unaffected by phospholipase A2 inhibitors, while reducing the effect of cytokines and arachadonate metabolic products produced following radiation exposure.
In this aspect, the present invention contemplates a pharmaceutical composition comprising an inhibitor of phospholipase A2 in a therapeutically effective amount and a physiologically acceptable carrier. The terms xe2x80x9ccontactedxe2x80x9d and xe2x80x9cexposed,xe2x80x9d when applied to a cell, are used herein to describe the process by which a phospholipase inhibitor is delivered to a target cell. Any method may be used to contact a cell with a phospholipase inhibitor, as long as the method results in decreased phospholipase A2 activity within the cell
The phrases xe2x80x9cpharmaceutically or pharmacologically acceptablexe2x80x9d refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a human. The appropriate doses for treating persons with radiation induced sequelae may be determined from a consideration of the condition to be treated and the properties of the composition being administered. This will be readily understood by those of skill in the art when in possession of the present disclosure.
A therapeutically effective amount of an inhibitor of phospholipase A2 that is combined with a carrier to produce a single dosage form varies depending upon the host treated and the particular mode of administration. A xe2x80x9ctherapeutically effective amountxe2x80x9d is an amount of a phospholipase inhibitor or similar agent that, when administered to an animal, is effective to reduce or eliminate phospholipase A2 activity within the animal.
As is well known in the art, a specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.
A composition of the present invention is typically administered orally or parenterally in dosage unit formulations containing standard, well known nontoxic physiologically acceptable carriers, adjuvants, and vehicles as desired. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intraarterial injection, or infusion techniques.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions are formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
Among the acceptable vehicles and solvents that may be employed are water, Ringer""s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Suitable doses for phospholipase A2 inhibitors would be for mepacrine (or quinacrine), 25 up to 200 mg every six hours for 5 doses prior to radiotherapy, and 200 mg once a day thereafter. As a further example, dosages of pentoxifylline range from 50 to 400 mg, given 3 times a day prior to and following radiotherapy. The skilled artisan is directed to xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d 15th Edition for dosages of various inhibitors. Some variation in dosage will necessarily-occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, preparations for human administration should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biologics standards.
As used herein, the term xe2x80x9cirradiated cellxe2x80x9d means a cell or tissue that has been exposed to an effective expression-inducing dose of ionizing radiation that stimulates or turns on a radiation responsive enhancer-promoter or causes a physiological response in the cell, and depends on the given cell type.
As used herein, the phrase xe2x80x9cenhancer-promoterxe2x80x9d means a composite unit that contains both enhancer and promoter elements. As used herein, a xe2x80x9cradiation responsive enhancer-promoterxe2x80x9d indicates an enhancer-promoter whose transcription controlling function is affected by ionizing radiation. Typically, upon exposure to an effective dose of ionizing radiation, a radiation responsive enhancer-promoter of the present invention stimulates or increases the rate of transcription of an encoding region controlled by that enhancer-promoter. An exemplary and preferred enhancer-promoter for use in a DNA molecule of the present invention is a CArG domain of an Egr-1 promoter, a promoter for tumor necrosis factor-alpha (TNF-xcex1) gene or a c-Jun promoter.
A radiation responsive enhancer-promoter is operatively linked to an encoding region that encodes at least one polypeptide. As used herein, the phrase xe2x80x9coperatively linkedxe2x80x9d means that an enhancer-promoter is connected to an encoding region in such a way that the transcription of that encoding region is controlled and regulated by that enhancer-promoter. Means for operatively linking an enhancer-promoter to an encoding region are well known in the art. As is also well known in the art, the precise orientation and location relative to an encoding region whose transcription is controlled, is dependent inter alia upon the specific nature of the enhancer-promoter. Thus, a TATA box minimal promoter is typically located from about 25 to about 30 base pairs upstream of a transcription initiation site and an upstream promoter element is typically located from about 100 to about 200 base pairs upstream of a transcription initiation site. In contrast, an enhancer can be located downstream from the initiation site and can be at a considerable distance from that site.
In one embodiment, an encoding region of a DNA molecule of the present invention encodes a single polypeptide. As used herein, the term xe2x80x9cpolypeptidexe2x80x9d means a polymer of amino acids connected by amide linkages, wherein the number of amino acid residues can range from about 5 to about one million. Preferably, a polypeptide has from about 10 to about 1000 amino acid residues and, even more preferably from about 20 to about 500 amino residues. Thus, as used herein, a polypeptide includes what is often referred to in the art as an oligopeptide (5-10 amino acid residues), a polypeptide (11-100 amino acid residues) and a protein ( greater than 100 amino acid residues). A polypeptide encoded by an encoding region can undergo post-translational modification to form conjugates with carbohydrates, lipids, nucleic acids and the like to form glycopolypeptides (e.g., glycoproteins), lipopolypeptides (e.g. lipoproteins) and other like conjugates.
Any polypeptide can be encoded by an encoding region of a DNA molecule of the present invention. An encoding region can comprise introns and exons so long as the encoding region comprises at least one open reading frame for transcription, translation and expression of that polypeptide. Thus, an encoding region can comprise a gene, a split gene or a cDNA molecule. In the event that the encoding region comprises a split gene (contains one or more introns), a cell transformed or transfected with a DNA molecule containing that split gene must have means for removing those introns and splicing together the exons in the RNA transcript from that DNA molecule if expression of that gene product is desired.
In a preferred embodiment an effective expression inducing amount is from about 2 to about 20 Gray (Gy) administered at a rate of from about 0.5 to about 2 Gy/minute. Even more preferably, an effective expression inducing amount of ionizing radiation is from about 5 to about 15 Gy.
As used herein, xe2x80x9cionizing radiationxe2x80x9d means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). An exemplary and preferred ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.
Cells containing a DNA molecule of the present invention encoding a particular polypeptide express that polypeptide when exposed to ionizing radiation.